TW202126287A - Use of composition containing curcumin and polyvinylpyrrolidone to combat biofilm of staphylococcus aureus for curing and/or preventing infectious bone diseases - Google Patents

Abstract

The present invention discloses use of a composition containing curcumin and polyvinylpyrrolidone (PVP) to combat the T cell immunosuppression and/or inflammatory response caused by a biofilm of staphylococcus aureus in an individual. Moreover, the individual has an infectious bone disease selected from the group consisting of osteitis, osteomyelitis, osteoarthritis, joint infections around the prosthesis, and a combination thereof; the biofilm is formed on the surface of a tissue selected from the group consisting of bone, bone marrow, joints, synovial sac, epithelium, and a combination thereof; the biofilm is formed on the surface of a prosthesis; and the prosthesis includes an artificial joint.

Description

Use a composition containing curcumin and polyvinylpyrrolidone to combat the biofilm of Staphylococcus aureus

The present invention relates to the use of a composition containing curcumin (curcumin, CUR) and polyvinylpyrrolidone (polyvinylpyrrolidone, PVP) to combat Staphylococcus aureus (biofilm) caused in a body T cell immunosuppression and/or inflammatory response.

Staphylococcus aureus is an opportunistic pathogen belonging to gram-positive cocci, which can colonize the skin and mucous membranes of humans and animals. membrane), and often cause nosocomial infections. For example, in orthopedic surgery (such as joint replacement), if it is on the patient’s wound or on the medical implants or prosthesis to be implanted Colonization of Staphylococcus aureus or its biofilm formation on the surface will cause the patient to suffer from infectious bone disease (osteitis), osteomyelitis (osteomyelitis), bone and joint Inflammation (osteoarthritis) and periprosthetic joint infections (PJIs).

At present, the clinical methods mainly used to combat Staphylococcus aureus include the administration of antibacterial agents [e.g., vancomycin], ultraviolet radiation and sterilization [e.g., high-pressure steam sterilization ( autoclaving) and flame sterilization]. However, the effects achieved by these methods are often not satisfactory. The main reason is that Staphylococcus aureus has the ability to form biofilms, which can protect the bacteria in the biofilms from ultraviolet light, high temperature or high pressure. Damage and increase the resistance of bacteria to host immune system attack and the toxic effect of antibiotics. For example, in Peng KT et al. (2017), PLoS One , 12(8):e0183271, Peng KT et al. established a mouse model of joint infection around the prosthesis by using Staphylococcus aureus biofilm, and through After the administration of vancomycin, it was found that vancomycin could not completely eliminate the infection of Staphylococcus aureus biofilm. In addition, Peng KT et al. further discovered that biofilms promote more bone marrow cells (BMC) to differentiate into monocytic myeloid derived suppressor cells (MDSC) instead of granules. Globular MDSC (granulocytic MDSC), mononuclear spherical MDSC will then be transformed into M2-macrophages (M2-macrophages) and act to inhibit the proliferation of T cells, in other words, biofilm can induce immunosuppressive effect (immunosuppressive effect) To resist the host's immune response.

Furthermore, even if the existing antibacterial methods completely eliminate the bacteria in the biofilm, the remaining components of the biofilm (especially the biofilm protein) will still trigger the host's inflammatory response. Therefore, relevant researchers in the field are dedicated to finding active components from traditional Chinese medicines (TCM) or plants that can effectively combat the biofilm formed by Staphylococcus aureus.

Curcumin (curcumin, CUR) is an active polyphenolic compound that can be isolated from the rhizome of turmeric (Curcuma longa). It is known that curcumin has anti-inflammatory, antioxidant, antibacterial, anticancer, and immunomodulation equivalent functions, so it has been widely used in the pharmaceutical industry and the food industry. superior.

Although curcumin has the above-mentioned beneficial effects, it still has the disadvantage of low water solubility. Low water solubility is an intrinsic property of many natural drug candidates, and is usually associated with bioavailability, thus limiting the development and clinical application of drugs. Therefore, many researchers are working to improve the water solubility of drug candidates. For example, in Yen FL et al . (2010), J. Agric. Food Chem. , 58:7376-7382, Yen FL et al. The nanoprecipitation method uses polyvinylpyrrolidone (PVP) as a hydrophilic carrier to prepare a composition containing curcumin and PVP. The composition has been confirmed through experiments that it can effectively increase the water solubility of curcumin, and has excellent antioxidant and antihepatoma activities.

In Tsekova P. et al. (2017), J. Mater. Sci. Mater. Med., doi:10.1007/s10856-017-6014-4, Tsekova P. et al. also used PVP to improve curcumin Water solubility, and prepared an electrospun сellulose acetate membranes modified with curcumin-PVP particles (curcumin-PVP particles), in which the curcumin-PVP particles are prepared by blending with acetone ( The curcumin in acetone) and PVP in water or ethanol are mixed to be prepared. The electrospun cellulose acetate membrane has been confirmed to have anti-Staphylococcus aureus activity through experiments.

After research, the applicant unexpectedly found that the composition containing curcumin and PVP not only has the effects of anti-oxidation, anti-liver cancer and anti-staphylococcus aureus, but also can effectively fight the biofilm formed by staphylococcus aureus in one body. The effect of T cell immunosuppression and/or inflammatory response caused by serotonin is expected to be used for the treatment and/or prevention of infectious bone disease.

Summary of the invention

Therefore, in the first aspect, the present invention provides a composition containing curcumin and polyvinylpyrrolidone for the preparation of a T cell immunosuppression and/ Or the use of inflammatory drugs.

In a second aspect, the present invention provides a method for combating T cell immunosuppression and/or inflammation caused by the biofilm of Staphylococcus aureus in an individual, which comprises administering to the individual a curcumin-containing drug And the composition of polyvinylpyrrolidone.

Detailed description of the invention

It should be understood that if any previous case publication is quoted here, the previous case publication does not constitute a recognition: in Taiwan or any other country, the previous case publication forms a common general in the art. Part of knowledge.

For the purpose of this specification, it will be clearly understood that the word "comprising" means "including but not limited to", and the word "comprises" has a corresponding meaning.

Unless otherwise defined, all technical and scientific terms used in this article have meanings commonly understood by those familiar with the art of the present invention. A person familiar with the art will recognize that many methods and materials similar or equivalent to those described herein can be used to implement the present invention. Of course, the present invention is by no means restricted by the described methods and materials.

In the development of drugs that can be used to combat the biofilm formed by Staphylococcus aureus (Staphylococcus aureus), the applicant found through experiments: a drug containing curcumin (curcumin, CUR) and polyvinylpyrrolidone (polyvinylpyrrolidone, The composition of PVP can effectively combat T cell immunosuppression and/or inflammatory response caused by the biofilm of Staphylococcus aureus, and is therefore expected to be used to treat and/or prevent infection Infectious bone disease.

Therefore, the present invention provides a composition containing curcumin and polyvinylpyrrolidone for the preparation of a pharmaceutical product used to combat T cell immunosuppression and/or inflammation caused by the biofilm of Staphylococcus aureus in one body the use of.

Preferably, the Staphylococcus aureus has resistance to antibiotics, and the antibiotic is selected from the group consisting of: β-lactam antibiotics [ For example, penicillin, methicillin, oxacillin, ampicillin, amoxicillin, faropenem, ertapenem , Ceftriaxone, and aztreonam, etc.], glycopeptides antibiotics [for example, vancomycin, teicoplanin, and Ramola Ning (ramoplanin, etc.), tetracycline antibiotics [e.g., tetracycline, doxycycline, oxytetracycline, chlortetracycline, and tigecycline ) Etc.], quinolone antibiotics [e.g., ciprofloxacin, norfloxacin, nalidixic acid, nemonoxacin, and Novobiocin (novobiocin, etc.), and their combinations. In a preferred embodiment of the present invention, the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA).

According to the present invention, the composition containing curcumin and polyvinylpyrrolidone can be prepared using a technique well known and commonly used by those skilled in the art. In this regard, you can refer to, for example, Yen FL et al . (2010) (same as above). Preferably, the composition containing curcumin and polyvinylpyrrolidone can be prepared by a method including the following steps: adding curcumin to ethanol to obtain an organic phase solution; Adding polyvinylpyrrolidone to water to obtain an aqueous phase solution; mixing the organic phase solution and the aqueous phase solution to obtain a mixture; performing a homogenization treatment on the mixture; And removing the ethanol in the homogenized mixture, thereby obtaining the composition containing curcumin and polyvinylpyrrolidone.

According to the present invention, the operating procedures, parameter conditions, etc. of the homogenization treatment fall within the scope of professionalism and routine technology of those who are familiar with the technology. Preferably, the homogenization treatment is performed at 5,000 to 25,000 rpm for 10 to 60 minutes. In a preferred embodiment of the present invention, the homogenization treatment is performed at 22,000 rpm for 25 minutes.

According to the present invention, curcumin and polyvinylpyrrolidone are in a ratio ranging from 1:1 (w/w) to 1:10 (w/w). In a preferred embodiment of the present invention, curcumin and polyvinylpyrrolidone are in a ratio of 1:6 (w/w).

As used herein, the term "biofilm" means a matrix of Staphylococcus aureus attached to a surface by secreting protective and adherent polymeric compounds. A slime layer or slime film is formed on the surface. Preferably, the biofilm contains biofilm protein. More preferably, the biofilm further comprises at least one of the following: polysaccharides, lipids, and extracellular nucleic acids.

Preferably, the biofilm does not substantially contain live bacteria.

As used herein, the term "substantially free of" means that a specified ingredient lacks a meaningful content, or does not contain the ingredient at all.

Preferably, the biofilm can be formed on the surface of a tissue selected from the following: bone, bone marrow, joint, synovial sac (bursa), epithelia (epithelia) , And their combination.

Preferably, the biofilm can be formed on the surface of a prosthesis. More preferably, the prosthesis includes artificial joints.

In a preferred embodiment of the present invention, the biofilm is formed on the femur.

According to the present invention, the inflammatory response is an inflammation of cells selected from the group consisting of bone marrow cells (BMC), chondrocytes, osteocytes, and tendon cells ( tenocyte), and their combination. In a preferred embodiment of the present invention, the inflammatory response is inflammation of bone marrow cells.

According to the present invention, the individual may have an infectious bone disease selected from the group consisting of: osteitis (osteitis), osteomyelitis (osteomyelitis), osteoarthritis (osteoarthritis), joint infections around the prosthesis (periprosthetic joint infections, PJIs), and their combinations. In a preferred embodiment of the present invention, the individual has a joint infection around the prosthesis.

According to the present invention, the medicine can be manufactured into a suitable parenteral administration using techniques well known to those skilled in the art.

According to the present invention, the drug may further include a pharmaceutically acceptable carrier that is widely used in drug manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more reagents selected from the group consisting of solvents, buffers, emulsifiers, suspending agents, decomposers ), disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent , Gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome and the like. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.

According to the present invention, the drug can be administered by a parenteral route selected from the group consisting of: intraperitoneal injection, intrapleural injection, intramuscular injection injection, intravenous injection, intraarterial injection, intraarticular injection, intrasynovial injection, intrathecal injection, intracranial injection injection, intraepidermal injection, subcutaneous injection, intradermal injection, intralesional injection, and sublingual administration.

According to the present invention, the medicine can be used in combination with one or more antibacterial agents selected from the group consisting of: vancomycin, rifamycin, and Rifampin, rifapentine, and rifaximin. In a preferred embodiment of the present invention, the medicine is used in combination with vancomycin.

The present invention also provides a method for combating T cell immunosuppression and/or inflammation caused by the biofilm of Staphylococcus aureus in an individual, which comprises administering to the individual a composition containing curcumin and polyvinylpyrrolidone Things.

As used herein, the terms "administering" and "administration" can be used interchangeably.

According to the present invention, the dosage and frequency of administration of the composition containing curcumin and polyvinylpyrrolidone will vary depending on the following factors: the severity of the disease to be improved, the route of administration, and the weight, age, and age of the individual to be improved Physical condition and reaction. The choice of dosage and frequency of administration falls within the scope of professionalism and routine techniques of those who are familiar with this technology.

Detailed description of the preferred embodiment

The present invention will be further described with respect to the following embodiments, but it should be understood that these embodiments are for illustrative purposes only, and should not be construed as limitations on the implementation of the present invention. Examples General experimental materials: 1. Curcumin (curcumin) and polyvinylpyrrolidone (PVP) used in the following examples are all purchased from Sigma-Aldrich. 2. Experimental animals:

The male C57BL/6J mice (C57BL/6J mice) (12 weeks old, weighing approximately 22 to 30 g) used in the following examples were purchased from the National Laboratory of Experimental Animal Center (National Laboratory Animal Center, ROC). All experimental animals were kept in an independent air-conditioned animal room with 12 hours of light and darkness, room temperature maintained at 25°C and relative humidity maintained at 50±10%, and water and feed were adequately supplied. All experimental procedures related to experimental animals are approved by the Institutional Animal Care and Use Committee of Chang Gung Memorial Hospital (Institutional Animal Care and Use Committee of Chang Gung Memorial Hospital) Care) ethical guidelines (ethical guidelines). General experimental methods: 1. Statistical analysis:

In the following examples, the experiment of each group is repeated 3 times, and the experimental data is represented by the standard error of the mean (SEM). All data are analyzed by one-way analysis of variance (ANOVA), followed by Tukey's post hoc test and GraphPad Prism software (GraphPad Software, San Diego, CA). To assess the differences between the groups. If the result of the statistical analysis is p <0.05, this indicates statistical significance. Example 1. Experimental method for preparing a composition containing curcumin and PVP:

The curcumin and PVP-containing compositions used in the following examples are mainly prepared according to the method described in Yen FL et al . (2010), J. Agric. Food Chem., 58: 7376-7382. In short, 50 mg of curcumin was mixed in 25 mL of ethanol to obtain an organic phase solution. In addition, 300 mg of PVP was mixed in 75 mL of pure water to obtain an aqueous phase solution. Next, the organic phase solution was immediately added to the aqueous phase solution and homogenization treatment was performed at 22,000 rpm for 25 minutes. Afterwards, the resulting mixture is subjected to rotary vacuum evaporation to remove ethanol, thereby obtaining a composition containing curcumin and PVP (hereinafter referred to as a CUR-PVP composition).

Afterwards, the CUR-PVP composition thus obtained was dissolved in phosphate-buffered saline (PBS) to prepare a stock solution of the CUR-PVP composition at a concentration of 6.25 mM. spare. Example 2. The composition contains curcumin and PVP in inhibiting S. aureus biofilm - induced inflammation of bone marrow cells [Staphylococcus aureus biofilm-induced inflammation of bone marrow cells (BMC)] to assess the effectiveness of the

In this example, the applicant analyzed the number of bone marrow-derived suppressor cells (myeloid derived suppressor cells, MDSC) and the levels of inflammatory cytokines and reactive oxygen species (ROS). To evaluate the effectiveness of a composition containing curcumin and PVP on Staphylococcus aureus biofilm-induced inflammation of bone marrow cells. Experimental materials: 1. Preparation of biofilm samples of Staphylococcus aureus:

First, it will be purchased from the Biosource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) in Taiwan (No. 331, Food Road, Hsinchu City, Taiwan) Staphylococcus aureus ATCC 43300 [it has methicillin-resistance] inoculated into brain heart infusion (BHI) medium (Bacto), and at a temperature The culture was carried out in an incubator set to 37°C for 4 days, and a biofilm was formed in the culture. After that, the culture was centrifuged at 3,900 xg for 15 minutes and the supernatant was removed, followed by autoclaving to sterilize the pellets, and then washed with PBS, followed by RPMI 1640 medium (added with 10% FBS) was re-dissolved. After that, use the BCA protein assay kit (Thermo Scientific) and use albumin (0-2,000 μg/mL) (Thermo Scientific) as the standard to perform biofilm protein analysis. Determination of concentration. Then, it was diluted with RPMI 1640 medium (with 10% FBS) to obtain a solution containing 0.2 mg/mL biofilm protein to be used as a biofilm sample. 2. Source and culture of bone marrow cells (BMC):

Refer to Liu X. and Quan N. et al. (2015), Bio. Protoc. , 5(20):e1631 to separate from the bone marrow of the femur of male C57BL/6J mice Bone marrow cells were extracted and supplemented with 10% FBS, 1 mM sodium pyruvate, 1% MEM non-essential amino acids (MEM NEAA), 20% penicillin- RPMI 1640 medium containing streptomycin and 0.05 mM β-mercaptoethanol (β-mercaptoethanol) was cultured (37°C, 5% CO ₂ ). The medium and its supplements were purchased from Thermo Fisher Scientific. 3. Preparation of curcumin stock solution:

The curcumin was dissolved in PBS to obtain a curcumin stock solution with a concentration of 6.25 mM. Experimental method: A. Pretreatment of composition containing curcumin and PVP and co-cultivation of Staphylococcus aureus biofilm:

First, divide the BMC cells obtained in item 2 of the above "experimental materials" into 1 normal control (normal control), 1 pathological control (pathological control), and 3 experimental groups (that is, experimental group 1). To 3) and 3 curcumin control groups (that is, curcumin control groups 1 to 3), the BMC cells of each group were ^{cultured in a quantity of 5×10 5} cells/well in 1,000 μL of RPMI 1640 medium (supplemented with 10% FBS) in a 24-well plate (24-well plate). Next, add the appropriate amount of CUR-PVP composition stock solution to experimental groups 1 to 3, so that the cell cultures of experimental groups 1 to 3 have final concentrations of 6.25, 12.5, and 25 μM CUR-PVP composition. And add an appropriate amount of the curcumin stock solution obtained in item 3 of the above "experimental materials" to the curcumin control group 1 to 3, so that the cell cultures of the curcumin control group 1 to 3 are respectively Curcumin with final concentrations of 6.25, 12.5 and 25 μM. As for the normal control group and the pathological control group, no treatment was done. After that, place a Transwell cell culture plate (polyester membrane with 0.4 μm holes) in each well of the 24-well culture plate, and then follow the above "experimental materials" The 0.2 mg/mL biofilm sample of Staphylococcus aureus obtained in item 1 of the above was added to the Transwell cell culture plate of the pathological control group, each experimental group, and each curcumin control group. As for the Transwell cell culture plate above the normal control group, the biofilm sample was not added. The cells of each group were cultured in an incubator (37°C, 5% CO ₂ ) for 48 hours. After that, the cell cultures of each group were used for the experiment in item B below. In addition, the cell cultures of the normal control group, the pathological control group, and the experimental groups 1 to 3 were used for the experiments of items C and D below. B. Determination of the relative number of MDSC cells:

First, the cell culture (1x10 ⁶ cells) of each group was collected and washed with PBS, and then FITC-CD11b antibody (Cat. No. 553310, BD Biosciences) (diluted 100 times with PBS) and PE-Gr-1 were added Antibody (Cat. No. 553128, BD Biosciences) (diluted 100-fold with PBS) and protected from light at 4°C for 30 minutes, followed by washing with PBS. After that, a FACSAria Fusion cell sorter (BD Biosciences, Franklin Lakes, NJ, USA) was used to collect MDSC cells in each group.

Next, BD FACSCanto II flow cytometer (BD FACSCanto II flow cytometer, BD Biosciences, USA) was used to measure the number of MDSC cells, and 10,000 cells were analyzed each time. The cells are excited by an argon ion laser beam of 488 nm to emit fluorescence, and the fluorescence intensity of FITC and PE is detected at wavelengths of 515-545 nm and 564-606 nm. The data obtained was analyzed by FACSDiva software. The relative number (multiple) of MDSC cells in each group is calculated by substituting the measured number of MDSC cells into the following formula (1): Formula (1) : A = B/C where: A = MDSC cells The relative number (multiple) B = the number of MDSC cells in each group C = the number of MDSC cells in the pathological control group

After that, analyze the obtained experimental data in accordance with the method described in item 1 "Statistical Analysis" of the "General Experimental Methods" above. C, interleukin -6 (interleukin-6, IL- 6), tumor necrosis factor -α (tumor necrosis factor-α, TNF-α) and monocyte chemoattractant protein -1 (monocyte chemoattractant protein-1, MCP -1) Determination of concentration:

First, the cell cultures of each group were collected, centrifuged at 2,000 rpm at 4°C for 2 minutes, and the supernatant was collected, and then the BD Cytometric Bead Array Mouse Inflammation Kit (BD Cytometric Bead Array Mouse Inflammation kit) (Cat. No. 552364, BD Biosciences) and determine the concentration of IL-6, TNF-α and MCP-1 according to the manufacturer's operating instructions.

After that, analyze the obtained experimental data in accordance with the method described in item 1 "Statistical Analysis" of the "General Experimental Methods" above. D. Determination of ROS output:

First, the cell cultures of each group were replaced with fresh medium, and then the appropriate amount of 2',7'-Dichlorofluorescin diacetate (2',7'-Dichlorofluorescin diacetate, DCFDA) (Cat. No. D6883, Sigma-Aldrich Co.) was added to the cell culture of each group so that each group had a final concentration of 20 μM DCFDA, and the light-proof reaction was performed at 25°C for 30 minutes. Then, the cell cultures of each group were collected and washed with PBS, and then analyzed with the BD FACSCanto II flow cytometer, and 10,000 cells were analyzed each time. The cells are excited by an argon ion laser beam of 488 nm to emit fluorescence, and the DCF fluorescence intensity of each group is detected at a wavelength of 515-545 nm.

After that, analyze the obtained experimental data in accordance with the method described in item 1 "Statistical Analysis" of the "General Experimental Methods" above. Results: A. Determination of the relative number of MDSC cells:

Figure 1 shows the relative number of MDSC cells measured in each group of cell cultures. It can be seen from Figure 1 that compared with the normal control group, the relative number of MDSC cells in the pathological control group has a significant increase, which means that the Staphylococcus aureus biofilm will cause the expansion of MDSC cells. Compared with the pathological control group, the relative number of MDSC cells in the curcumin control group 1 to 3 did not change significantly. As for the relative number of MDSC cells in the experimental group 1 to 3, there was a significant decrease. Develop a dose-dependent effect. The results of this experiment show that, compared to curcumin, the composition containing curcumin and PVP has higher water solubility and can effectively inhibit Staphylococcus aureus biofilm-induced MDSC cell expansion. B. Determination of the concentration of IL-6 , TNF-α and MCP-1:

Figures 2 to 4 respectively show the concentrations of IL-6, TNF-α and MCP-1 measured in the cell cultures of each group. It can be seen from Figure 2 to Figure 4 that compared with the normal control group, the cells of the pathological control group will express IL-6, TNF-α and MCP-1 in a large amount, which indicates that the Staphylococcus aureus biofilm successfully induces BMC The cells produce an inflammatory response. Compared with the cells in the pathological control group, the concentrations of IL-6, TNF-α, and MCP-1 in the cells in the experimental groups 1 to 3 were significantly reduced, and showed a dose-dependent effect. The results of this experiment show that the composition containing curcumin and PVP can effectively protect cells against Staphylococcus aureus biofilm-induced inflammation. C. Determination of ROS output:

Figure 5 shows the measured DCF fluorescence intensity of each group of cell cultures. It can be seen from Figure 5 that compared with the normal control group, the measured DCF fluorescence intensity of the cells in the pathological control group has a significant increase, which indicates that the Staphylococcus aureus biofilm successfully induced the BMC cells to produce oxidative stress. Compared with the cells in the pathological control group, the DCF fluorescence intensity measured by the cells in the experimental groups 1 to 3 has a significant decrease, and exhibits a dose-dependent effect. The results of this experiment show that the composition containing curcumin and PVP can effectively protect cells against Staphylococcus aureus biofilm-induced oxidative stress.

Based on the above experimental results, it can be seen that the composition containing curcumin and PVP can effectively inhibit Staphylococcus aureus biofilm-induced inflammation of bone marrow cells. In the following examples, the applicant then explored whether the composition can further prevent immune disorders caused by Staphylococcus aureus biofilm. Embodiment 3. The composition contains curcumin and PVP for S. aureus biofilm Example - Effect of immunosuppressive effects induced MDSC cells (immunosuppressive effect) of

In this example, the applicant used the CUR-PVP composition obtained in Example 1 above to treat MDSC cells induced by Staphylococcus aureus biofilm, and then collected the MDSC cells and used them with T The cells were co-cultured to evaluate the effect of the CUR-PVP composition on the immunosuppressive effect of Staphylococcus aureus biofilm-induced MDSC cells. Experimental materials: 1. Source and culture of T cells:

With reference to the method described in Wohler JE and Barnum SR et al. (2009), Mol. Immunol. , 46: 1007-1010 and by using a nylon wool column (nylon wool column), from male C57BL/6J mice T cells were isolated from the spleen and cultured in RPMI 1640 medium supplemented with 10% FBS, 1 mM sodium pyruvate, 1% MEM NEAA, 20% penicillin-streptomycin, and 0.05 mM β-mercaptoethanol (37 ℃, 5% CO ₂ ).

Next, in order to facilitate the observation of T cells, perform fluorescent labeling according to the following steps: add an appropriate amount of 5(6)-carboxyfluorescein N-hydroxysuccinimide ester [5(6)- carboxyfluorescein N-hydroxysuccinimidyl ester, CFSE] (BD Biosciences, Cat. No. 565082) and mix well, and then incubate in an incubator (37℃, 5% CO ₂ ) for 10 minutes, so that T cells are labeled With CFSE, it will show green fluorescence. experimental method:

First, divide the BMC cells obtained in item 2 of the "experimental materials" in Example 2 above into 1 normal control group, 1 pathological control group, and 2 sterile control groups (that is, sterile control groups 1 and 2). ) And two experimental groups (ie, experimental groups 1 and 2). BMC cells in each group were cultured in 24 cells containing 1,000 μL of RPMI 1640 medium (supplemented with 10% FBS) ^{at a rate of 5×10 5 cells/well.} -Well in the culture dish. Next, an appropriate amount of the stock solution of the CUR-PVP composition was added to the sterile control group 1 and the experimental group 1 respectively, so that the cell cultures of the sterile control group 1 and the experimental group 1 had a final concentration of 6.25 μM CUR- The PVP composition and the stock solution of the appropriate amount of CUR-PVP composition were added to the sterile control group 2 and the experimental group 2 respectively, so that the cell cultures of the sterile control group 2 and the experimental group 2 had a final concentration of 12.5 μM The composition of CUR-PVP. As for the normal control group and the pathological control group, no treatment was done. After that, place a Transwell cell culture plate in each well of the 24-well culture plate, and then add the 0.2 mg/mL Staphylococcus aureus obtained in item 1 of the "Experimental Materials" of Example 2 above. Biofilm samples were added to the Transwell cell culture dishes of the pathological control group and each experimental group. As for the normal control group and the Transwell cell culture plate above each sterile control group, the biofilm sample was not added. The cells of each group were cultured in an incubator (37°C, 5% CO ₂ ) for 48 hours.

After that, according to the method described in item B of the "experimental method" of Example 2, the cell cultures of each group were collected for MDSC cells.

Next, combine the MDSC cells collected in each group with the CFSE-labeled T cells obtained in item 1 of the above "Experimental Materials" in a ratio of 0.25:1 to the number of cells (the total number of cells is 2x10). ⁵ cells/well) were co-cultured in a 96-well culture dish containing 200 μL of RPMI 1640 medium (supplemented with 10% FBS), and then Dynabeads ^® Mouse T-Activator CD3/CD28 (Thermo Fisher Scientific) was used according to the manufacturer The operation guide for T cell activation (activation) lasted 48 hours. In addition, T cells that were not co-cultured with MDSC cells were used as controls.

After that, the cell cultures of each group were collected and the number of T cells was measured with a BD FACSCanto II flow cytometer, and 10,000 cells were analyzed each time. The cells are excited by an argon ion laser beam of 488 nm to emit fluorescence, and the fluorescence intensity is detected at a wavelength of 515-545 nm. The data obtained was analyzed by FACSDiva software. The T cell proliferation percentage (%) of each group is calculated by substituting the measured number of T cells into the following formula (2): formula (2) : D = (E/F) × 100 where: D = T cell proliferation percentage (%) E = the number of T cells in each group F = the number of control T cells

After that, analyze the obtained experimental data in accordance with the method described in item 1 "Statistical Analysis" of the "General Experimental Methods" above. result:

Figure 6 shows the percentage of T cell proliferation measured in cell cultures of each group. It can be seen from Figure 6 that compared with the normal control group, the percentage of T cell proliferation in the pathological control group is significantly reduced, which means that the Staphylococcus aureus biofilm will induce more monocytic MDSC cells and inhibit T cells. Hyperplasia. Compared with the pathological control group, the percentage of T cell proliferation in experimental groups 1 and 2 both increased significantly, and showed a dose-dependent effect. In particular, the percentage of T cell proliferation in the experimental group 2 was even similar to that of the sterile control group 2. The results of this experiment show that the composition containing curcumin and PVP can effectively protect T cells from the immunosuppressive effects induced by Staphylococcus aureus biofilm. Example 4. Evaluation of the effectiveness of a composition containing curcumin and PVP in the treatment of periprosthetic joint infections (PJIs)

In this example, the applicant used the biofilm formed by Staphylococcus aureus to induce joint infections around the prosthesis in mice, and through the appearance observation, histopathological analysis and peripheral blood of the mouse femur (femur). The number of MDSC cells in peripheral blood) was measured to evaluate the therapeutic effect of the composition containing curcumin and PVP on the treatment of joint infections around the prosthesis. Experimental method: A. Induction of joint infection around prosthesis and administration of CUR-PVP composition:

Male C57BL/6J mice were randomly divided into 6 groups (n=4 in each group), including 1 sham-operated group, 1 pathology control group, 1 antibiotic group, and 3 experimental groups ( That is, experimental group 1 to 3). Mice in each group were anesthetized with a mixture containing 50% zoletil and 2% rompun. Next, the skin adjacent to the right knee of each group of mice was disinfected with povidone-iodine, and then the skin was cut with a surgical knife to form an incision, and then a 26 -26-gauge needle to form a burr hole extending from the femoral intercondylar notch to the intramedullary canal, and then a length of 0.5 cm and diameter A 0.6 mm kirschner wire (Synthes GmbH, Solothurn, Switzerland) was implanted in the intramedullary canal. After that, an appropriate amount of Staphylococcus aureus biofilm sample (the bacterial concentration of Staphylococcus aureus is about 2x10 ³ CFU) was inoculated into the intramedullary canal of the pathological control group, antibiotic group and each experimental group to induce the prosthesis Infection of surrounding joints. As for the sham operation group, no biofilm samples of Staphylococcus aureus were inoculated.

Next, bone wax was used to seal the drill holes of each group of mice and Vicryl 4-0 sutures were used to suture the wound. After that, mice in the antibiotic group were immediately injected with vancomycin (purchased from Sandoz) (dose 2 mg/kg) by intraperitoneal injection, while mice in experimental groups 1 to 3 were injected In addition to vancomycin, CUR-PVP composition was injected intraperitoneally at different doses (5 mg/kg, 10 mg/kg and 20 mg/kg). As for the mice in the sham operation group and the pathological control group, they were injected intraperitoneally with normal saline (dose of 1 mL/kg). The mice in each group were administered once a day for 7 days.

At the end of the second week after the start of the administration, peripheral blood was collected from each group of mice, and then sacrificed and the femurs were taken out. The femur and peripheral blood thus obtained were used for experiments in items B to D below. B. Observation of the appearance of the femur:

Use a micro-computed tomography (μ-CT) system (model SKYSCAN 1076, brand name SKYSCAN) to take pictures of the femurs of each group of mice obtained in item A above, Then observe its appearance. C. Histopathological analysis of femur:

After taking pictures of the femurs of each group of mice, 10% formalin was used for fixation. Then, the fixed femur was embedded with paraffin, and then sliced, thereby obtaining tissue sections with a thickness of 3 μm.

After that, the obtained tissue section was stained by using hematoxylin-eosin (hematoxylin-eosin) and according to the well-known and customary technique of those skilled in the art, and then using the Motic Easyscan digital slide scanner (Motic Easyscan Digital Slide Scanner) (purchased from Motic Hong Kong Limited, Hong Kong, China) and DSAssistant and EasyScanner software to scan (resolution 0.26 μm/pixel) and take pictures (magnification 6.5 times), and then observe whether there is Inflammation symptoms appeared, and the severity of the inflammation symptoms was evaluated according to the method described in Rigoni R. et al . (2016), J. Exp. Med. , 213:355-375, and the inflammation of each group of mice was obtained Index score (inflammatory index score).

After that, analyze the obtained experimental data in accordance with the method described in item 1 "Statistical Analysis" of the "General Experimental Methods" above. D. Determination of MDSC cells in peripheral blood:

Take 50 μL of the peripheral blood of each group of mice obtained in item A above and use ACK lysis buffer to dissolve red blood cells, and then PBS (with 2% FBS) to adequately suspend the remaining white blood cells. Next, the collection of MDSC cells and the determination of the number of MDSC cells were performed in accordance with the method described in Item B of the "Experimental Method" of Example 2.

After that, analyze the obtained experimental data in accordance with the method described in item 1 "Statistical Analysis" of the "General Experimental Methods" above. Results: A. Observation of the appearance of the femur:

Fig. 7 shows the photos of the femurs of mice in each group taken by the micro-computer tomography system at the end of the second week after the start of the administration. It can be seen from Figure 7 that compared with the sham operation group, the femurs of the mice in the pathological control group had severe osteolytic bone destruction and pathological fracture, which indicated golden yellow Staphylococcal biofilm successfully induced joint infections around the prosthesis in mice. Compared with the pathological control group, the osteolytic bone destruction and pathological fractures observed in the femurs of the mice in the antibiotic group were only slightly improved. As for the experimental groups 1 to 3, significant improvements were observed. The improvement, and shows a dose-dependent effect. The results of this experiment show that the composition containing curcumin and PVP can effectively enhance the effectiveness of antibiotics in the treatment of joint infections around the prosthesis and improve bone destruction. B. Histopathological analysis of femur:

Fig. 8 shows the inflammatory index scores observed in the femoral tissue of each group of mice by staining with hematoxylin and eosin at the end of the second week after the start of the administration. It can be seen from Figure 8 that compared with the sham operation group, the inflammation index score value of the mice in the pathological control group has a significant increase, which means that the Staphylococcus aureus biofilm will induce inflammation in the mice. Compared with the pathological control group, the inflammatory index scores of the mice in the antibiotic group and the experimental groups 1 to 3 have decreased, and the inflammatory index scores of the mice in the experimental group 2 and the experimental group 3 Compared with those in the antibiotic group, there was a significant decrease. The results of this experiment show that the composition containing curcumin and PVP can effectively enhance the effectiveness of antibiotics in the treatment of joint infections around the prosthesis and improve bone inflammation. C. Determination of MDSC cells in peripheral blood:

Figure 9 shows the percentage of the number of MDSC cells in the peripheral blood measured by the mice in each group at the end of the second week after the start of the administration. It can be seen from Figure 9 that compared with the sham operation group, the percentage of the number of MDSC cells in the peripheral blood of the mice in the pathological control group has a significant increase, which indicates that the Staphylococcus aureus biofilm successfully induced the peripheral blood of the mice MDSC amplification in. Compared with the pathological control group, the percentage of the number of MDSC cells in the peripheral blood of the mice in the antibiotic group and the experimental groups 1 to 3 decreased. In particular, the percentage of the number of MDSC cells in the peripheral blood of the mice in the experimental group 3 was significantly lower than that in the antibiotic group. The results of this experiment show that the composition containing curcumin and PVP can effectively enhance the effectiveness of antibiotics in the treatment of joint infections around the prosthesis and improve immune disorders.

Based on the above experimental results, the applicant believes that the composition containing curcumin and PVP can be used to treat infectious bone disease induced by Staphylococcus aureus, especially the joint infection around the prosthesis.

All patents and documents cited in this specification are incorporated into this case as reference materials in their entirety. If there is a conflict, the detailed description of the case (including definitions) will prevail.

Although the present invention has been described with reference to the above specific specific examples, it is obvious that many modifications and changes can be made without departing from the scope and spirit of the present invention. Therefore, it is intended that the present invention is only limited by the scope of the patent application attached hereto.

The above and other objects, features and advantages of the present invention will become apparent with reference to the following detailed description and preferred embodiments and the accompanying drawings, in which: Figure 1 shows the measured results of each group of cell cultures The relative number of MDSC cells obtained, in which the normal control group represents the cells without any treatment; the pathological control group represents the cells that have been treated with 0.2 mg/mL Staphylococcus aureus biofilm samples; the experimental group 1 represents the cells that have been treated with 0.2 mg/mL Staphylococcus aureus. mg/mL Staphylococcus aureus biofilm samples and cells with 6.25 µM CUR-PVP composition; experimental group 2 represents the biofilm samples treated with 0.2 mg/mL Staphylococcus aureus and 12.5 µM CUR-PVP composition Cells; Experimental group 3 represents cells treated with 0.2 mg/mL Staphylococcus aureus biofilm samples and 25 µM CUR-PVP composition; Curcumin control group 1 represents cells treated with 0.2 mg/mL Staphylococcus aureus Biofilm samples and 6.25 µM curcumin cells; Curcumin control group 2 means biofilm samples treated with 0.2 mg/mL Staphylococcus aureus and 12.5 µM curcumin cells; curcumin control group 3 means cells treated with 0.2 mg/mL Staphylococcus aureus mg/mL Staphylococcus aureus biofilm samples and 25 µM curcumin cells; "*" means when compared with the pathological control group, p <0.05; and "**" means when compared with the pathological control group, p <0.01; Figures 2 to 4 respectively show the concentrations of IL-6, TNF-α and MCP-1 measured in the cell cultures of each group. The normal control group represents the cells without any treatment; the pathological control group represents the Cells treated with 0.2 mg/mL Staphylococcus aureus biofilm samples; experimental group 1 represents cells treated with 0.2 mg/mL Staphylococcus aureus biofilm samples and 6.25 µM CUR-PVP composition; experimental group 2 It means that the biofilm sample of 0.2 mg/mL Staphylococcus aureus and 12.5 µM CUR-PVP composition is processed; the experimental group 3 means that the biofilm sample of 0.2 mg/mL Staphylococcus aureus and 25 µM CUR are processed. -PVP composition cells; "*" means when compared with the pathological control group, p <0.05; and "**" means when compared with the pathological control group, p <0.01; Figure 5 shows the cell culture of each group The measured DCF fluorescence intensity, where the normal control group represents the cells without any treatment; the pathological control group represents the cells that have been treated with 0.2 mg/mL Staphylococcus aureus biofilm samples; the experimental group 1 represents the cells that have been treated with 0.2 mg/mL Staphylococcus aureus. mg/mL Staphylococcus aureus biofilm samples and 6.25 µM CUR-PVP composition cells; experimental group 2 represents the biofilm samples treated with 0.2 mg/mL Staphylococcus aureus and 12.5 µM CUR-PVP composition cells; experimental group 3 represents a biofilm sample treated with 0.2 mg/mL Staphylococcus aureus and 25 µM CUR-PVP composition cells; and “**” represents when compared with the pathological control group For comparison, p <0.01; Figure 6 shows the percentage of T cell proliferation measured by cell cultures in each group, where "**" means that when compared with the pathological control group, p <0.01; Figure 7 shows the percentage of T cell proliferation after the start of administration At the end of the second week, the femurs of the mice in each group were taken by the micro-computer tomography system. The sham operation group represents the mice without any treatment; the pathological control group represents the treatment with 2x10 ³ CFU Mice with biofilm samples of Staphylococcus aureus; antibiotic group means mice treated with 2x10 ³ CFU biofilm samples of Staphylococcus aureus and vancomycin (dose 2 mg/kg); experimental group 1 means mice Mice treated with 2x10 ³ CFU of Staphylococcus aureus biofilm samples and CUR-PVP composition (dose 5 mg/kg); experimental group 2 represents 2x10 ³ CFU of Staphylococcus aureus biofilm samples and CUR-PVP composition (dose of 10 mg/kg) mice; and experimental group 3 represents ^{a biofilm sample treated with 2x10 3} CFU of Staphylococcus aureus and CUR-PVP composition (dose of 20 mg/kg) Figure 8 shows the inflammatory index scores observed in the femoral tissue of each group of mice by staining with hematoxylin and eosin at the end of the second week after the start of the administration, and the sham operation group Means the mice without any treatment; the pathological control group means the mice treated with 2x10 ³ CFU Staphylococcus aureus biofilm samples; the antibiotic group means the mice treated with 2×10 ³ CFU Staphylococcus aureus biofilm samples and Vango Mice with a dose of 2 mg/kg of mycin; Experimental group 1 represents mice treated with ^{a biofilm sample of 2x10 3} CFU of Staphylococcus aureus and a CUR-PVP composition (dose of 5 mg/kg); Experimental group 2 represents mice treated with 2x10 ³ CFU Staphylococcus aureus biofilm samples and CUR-PVP composition (dose 10 mg/kg); experimental group 3 represents mice treated with 2x10 ³ CFU Staphylococcus aureus "*" means when compared with the antibiotic group, p <0.05; and "**" means when compared with the antibiotic group, p <0.01; and Figure 9 shows the percentage of the number of MDSC cells in the peripheral blood measured by the mice in each group at the end of the second week after the start of the administration, where the sham operation group represents the mice without any treatment; The pathological control group represents ^{a biofilm sample processed with 2x10 3} CFU of Staphylococcus aureus The antibiotic group refers to the mice that were treated with 2x10 ³ CFU of Staphylococcus aureus biofilm samples and vancomycin (dose 2 mg/kg); the experimental group 1 refers to the 2x10 ³ CFU golden grapes Biofilm samples of cocci and mice with CUR-PVP composition (dose of 5 mg/kg); experimental group 2 represents ^{a biofilm sample of 2x10 3} CFU of Staphylococcus aureus and CUR-PVP composition (dose of 10 mg/kg) mice; experimental group 3 means mice treated with 2x10 ³ CFU of Staphylococcus aureus biofilm samples and CUR-PVP composition (dose 20 mg/kg); and "*" means When compared with the antibiotic group, p <0.05.

Claims (8)

A composition containing curcumin and polyvinylpyrrolidone is supplied for the preparation of a medicine for the preparation of a medicine used to fight against T cell immunosuppression and/or inflammation caused by the biofilm of Staphylococcus aureus in one body. The use according to claim 1, wherein the individual has an infectious bone disease selected from the group consisting of osteitis, osteomyelitis, osteoarthritis, joint infections around the prosthesis, and combinations thereof. The use according to claim 1, wherein the biofilm is formed on the surface of a tissue selected from the group consisting of bone, bone marrow, joints, synovial sac, epithelium, and combinations thereof. The use of claim 1, wherein the biofilm is formed on the surface of a prosthesis. The use of claim 4, wherein the prosthesis includes an artificial joint. Such as the use of claim 1, wherein the biofilm does not substantially contain live bacteria. Such as the use of claim 1, wherein the drug is in a dosage form for parenteral administration. Such as the use of claim 1, wherein the medicine can be used in combination with one or more antibacterial agents selected from the group consisting of: vancomycin, rapamycin, rifampicin, rifapentin Tine, and rifaximin.

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